U.S. patent number 7,811,758 [Application Number 11/547,093] was granted by the patent office on 2010-10-12 for method and apparatus of automatically isolating and purifying nucleic acid.
This patent grant is currently assigned to Fujifilm Corporation. Invention is credited to Yoshihiko Makino, Toshihiro Mori.
United States Patent |
7,811,758 |
Mori , et al. |
October 12, 2010 |
Method and apparatus of automatically isolating and purifying
nucleic acid
Abstract
A method of automatically isolating and purifying nucleic acid
from a nucleic acid-containing specimen is provided, the method
comprising: injecting a liquid into a cartridge for isolation and
purification of a nucleic acid including at least two openings from
one opening of the at least two openings, in which the cartridge
includes a container having the at least two openings and
containing a nucleic acid-adsorbent solid phase; passing the liquid
through the nucleic acid-adsorbent solid phase by a pressure
difference generated by a pressure generation means for generating
a pressure difference between the inside and outside of the
container; and discharging the liquid from the other opening of the
container to the outside of the container by a pressure difference
generated by the pressure generation means, wherein a pressure
generated in the inside of the container by the pressure generation
means is measured, a pressure change velocity and a pressure change
acceleration are calculated on the basis of the value of the
measured pressure, and the timing of completion of discharge of the
liquid from the container is determined by use of a temporal change
pattern of at least one of the measured pressure, the pressure
change velocity and the pressure change acceleration.
Inventors: |
Mori; Toshihiro (Asaka,
JP), Makino; Yoshihiko (Asaka, JP) |
Assignee: |
Fujifilm Corporation (Tokyo,
JP)
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Family
ID: |
35056190 |
Appl.
No.: |
11/547,093 |
Filed: |
March 28, 2005 |
PCT
Filed: |
March 28, 2005 |
PCT No.: |
PCT/JP2005/006530 |
371(c)(1),(2),(4) Date: |
September 29, 2006 |
PCT
Pub. No.: |
WO2005/093053 |
PCT
Pub. Date: |
October 06, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20080275228 A1 |
Nov 6, 2008 |
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Foreign Application Priority Data
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Mar 29, 2004 [JP] |
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2004-094396 |
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Current U.S.
Class: |
435/6.1;
435/6.18; 536/25.4; 435/283.1; 536/22.1 |
Current CPC
Class: |
B01D
15/163 (20130101); B01J 20/265 (20130101); B01J
20/28054 (20130101); B01D 15/14 (20130101); B01J
20/26 (20130101); B01J 20/28033 (20130101); B01J
20/262 (20130101); B01J 20/28014 (20130101); C12N
15/1017 (20130101); C12N 15/1006 (20130101); G01N
30/32 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12M 1/34 (20060101); C07H
21/00 (20060101) |
Field of
Search: |
;435/6,283.1
;536/22.1,25.4 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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19 33 846 |
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Jan 1970 |
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DE |
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7-51065 |
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Jun 1995 |
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JP |
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9-179603 |
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Jul 1997 |
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JP |
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10-19883 |
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Jan 1998 |
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JP |
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11-333258 |
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Dec 1999 |
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JP |
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2003-128691 |
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May 2003 |
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JP |
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WO-89/09265 |
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Oct 1989 |
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WO |
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WO-97/26532 |
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Jul 1997 |
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WO |
|
Primary Examiner: Kapushoc; Stephen
Assistant Examiner: Bhat; Narayan K
Attorney, Agent or Firm: Birch, Stewart, Kolasch &
Birch, LLP
Claims
The invention claimed is:
1. A method of automatically isolating and purifying nucleic acid
from a nucleic acid-containing specimen, the method comprises: a)
injecting a liquid into a cartridge for isolation and purification
of a nucleic acid including at least two openings from one opening
of the at least two openings, in which the cartridge includes a
container having the at least two openings and containing a nucleic
acid-adsorbent solid phase; b) passing the liquid through the
nucleic acid-adsorbent solid phase by a pressure difference
generated by a pressure generation means for generating a pressure
difference between the inside and outside of the container; wherein
a pressure generated in the inside of the container by the pressure
generation means is measured and a pressure change velocity and a
pressure change acceleration are calculated on the basis of the
value of the measured pressure, and c) discharging the liquid from
the other opening of the container to the outside of the container
by a pressure difference generated by the pressure generation
means, wherein the timing of completion of discharge of the liquid
from the container is determined by use of a temporal change
pattern of a measured pressure selected from the group consisting
of (1) when the pressure of the inside of the container reaches a
set value in the middle of pressurizing the inside of the cartridge
by the pressure generation means, the pressurization by the
pressure generation means is stopped, and a point of time when the
pressure change acceleration in said container is minimized after
the stop of pressurization is determined as the timing of
completion of discharge of the liquid from the container; (2) when
the pressure of the inside of the container does not reach a set
value in the middle of pressurizing the inside of the cartridge by
the pressure generation means, at least one of a point of time when
the pressure in the container is maximized and the pressure change
velocity in the container becomes zero or less and a point of time
when the pressure in the container is maximized and the pressure
change acceleration in the container is minimized, is determined as
the timing of completion of discharge of the liquid from the
container; and (3) when the pressure of the inside of the container
does not reach a set value and is not maximized in the middle of
pressurizing the inside of the cartridge by the pressure generation
means, a point of time when the pressure change velocity in the
container becomes lower than a threshold value, is determined as
the timing of completion of discharge of the liquid from the
container.
2. The method according to claim 1, which comprises: (1) injecting
and pressurizing a nucleic acid-containing specimen liquid into the
container through one of the at least two openings, and discharging
the nucleic acid-containing specimen liquid from the other opening
to thereby adsorb a nucleic acid contained in the nucleic
acid-containing specimen liquid to the nucleic acid-adsorbent solid
phase; (2) dispensing and pressurizing a washing liquid into the
container through one of the at least two openings, and discharging
the washing liquid from the container through the other opening to
remove impurities; and (3) dispensing and pressurizing an elution
liquid into the container through one of the at least two openings
to isolate and discharge the nucleic acid adsorbed to the nucleic
acid-adsorbent solid phase from the container through the other
opening together with the elution liquid to thereby collect nucleic
acid.
3. The method according to claim 2, wherein in response of the
determined timing of completion of discharge of the liquid, the
subsequent step is performed.
Description
TECHNICAL FIELD
The present invention relates to a method of automatically
isolating and purifying nucleic acid and apparatus for
automatically isolating and purifying nucleic acid. More
particularly it relates to a technique for determining a completion
of movement of a liquid in a process of moving the liquid by a
pressure difference generated by a pressure generation means.
BACKGROUND ART
As a popularly known nucleic acid isolation and purification
method, there is a method for adsorbing nucleic acid on a solid
phase of silicon dioxide, silica polymer, magnesium silicate or the
like and isolating and purifying nucleic acid by a succeeding
operation of washing, desorption, and so on (e.g. Patent Document
1). This method is excellent in isolating performance but is
insufficient in simplicity, rapidity, automation and
miniaturization adaptability. There are problems in industrial
difficulty in mass production of adsorbent media having the same
performance, inconvenience in handling, difficulty in processing
the adsorbent media to various shapes, and so on.
As a method for isolating and purifying nucleic acid easily and
efficiently to solve the aforementioned problems, there has been
disclosed a method in which a solution for adsorbing nucleic acid
on a porous film and a solution for desorbing nucleic acid from the
porous film are used, and nucleic acid is adsorbed/desorbed on/from
a porous film made of an organic macromolecule having a hydroxyl
group on its surface to thereby automatically isolate and purify
nucleic acid on the oasis of pressurizing (Patent Document 2).
Concerning a pressurizing apparatus for supplying pressurized air
to a isolator having a filter in its inside, there have been
disclosed a pressurized air supply apparatus, a pressure sensor and
an apparatus for stopping pressurizing when pressure detected by
the pressure sensor becomes a set value or higher (Patent Document
3).
[Patent Document 1] Japanese Patent Publication No. 51065/1995
[Patent Document 2] Japanese Patent Laid-Open No. 2003-128691
[Patent Document 3] Japanese Patent Laid-Open No. 19883/1998
DISCLOSURE OF THE INVENTION
In Patent Document 1, there is however no description about a
specific method for finding the timing of completion of each of the
steps for purifying nucleic acid. In Patent Document 2, there is no
description about algorithm for determining a point of time of
completion of each step though the apparatus described in Patent
Document 2 stops pressurizing and controls pressure to prevent
hematolysis because hematolysis occurs when too high pressure is
applied at the time of isolation of hematocytes. Moreover, even
when this technique is used, it is practically impossible to
determine a point of time of completion of each step before and
after a point of set pressure because the time required for passage
of the liquid and pressure and patterns of pressure change required
for isolation vary according to the individual difference of the
isolator having the filter in its inside, the kind of the filter,
the individual difference due to the production lot of the filter,
the difference in kind of the liquid to be used, and so on.
As described above, in the step of discharging the liquid injected
into the container from the container by pressurizing, it is
difficult to automatically determine the point of time of
completion of discharge of the liquid accurately and rapidly
because the time required for completion of discharge, and pressure
and patterns of pressure change required for isolation vary
according to the difference in kind of the nucleic acid-adsorbent
solid phase stored in the container, the difference in production
lot of the nucleic acid-adsorbent solid phase, the difference in
production lot of the container per se, the difference in the
container in each production lot, the kind of the liquid to be
used, and so on.
The present invention is designed in consideration of the problems
in the background art, and an object of the invention is to provide
an method of automatically isolating and purifying nucleic acid and
the apparatus, in which completion of discharge of a liquid can be
automatically determined accurately and rapidly when the liquid is
injected into the container having the nucleic acid-adsorbent solid
phase stored therein and is discharged from the container through
the nucleic acid-adsorbent solid phase on the basis of the pressure
difference between the inside and outside of the container.
The present inventors have made eager examination to solve the
aforementioned problems. As a result, it has been found that in the
step of injecting a liquid into a container having a nucleic
acid-adsorbent solid phase and two openings through one opening of
the two openings and then pressurized and discharged from the
container through the other opening, a completion of discharge of
the liquid can be determined automatically by detecting patterns of
temporal change of pressure parameters in a container in a
combination by a pressure sensor in the middle of change of
pressure. Thus, the invention is accomplished.
That is, the invention is configured as follows.
1. A method of automatically isolating and purifying nucleic acid
from a nucleic acid-containing specimen, the method comprises:
injecting a liquid into a cartridge for isolation and purification
of a nucleic acid including at least two openings from one opening
of the at least two openings, in which the cartridge includes a
container having the at least two openings and containing a nucleic
acid-adsorbent solid phase;
passing the liquid through the nucleic acid-adsorbent solid phase
by a pressure difference generated by a pressure generation means
for generating a pressure difference between the inside and outside
of the container; and
discharging the liquid from the other opening of the container to
the outside of the container by a pressure difference generated by
the pressure generation means,
wherein a pressure generated in the inside of the container by the
pressure generation means is measured,
a pressure change velocity and a pressure change acceleration are
calculated on the basis of the value of the measured pressure,
and
the timing of completion of discharge of the liquid from the
container is determined by use of a temporal change pattern of at
least one of the measured pressure, the pressure change velocity
and the pressure change acceleration.
2. The method according to the item 1, wherein, when the pressure
of the inside of the container reaches a set value in the middle of
pressurizing the inside of the cartridge by the pressure generation
means, the pressurization by the pressure generation means is
stopped, and a point of time when the pressure change acceleration
in said container is minimized after the stop of pressurization is
determined as the timing of completion of discharge of the liquid
from the container.
3. The method according to the item 1 or 2, wherein, when the
pressure of the inside of the container does not reach a set value
in the middle of pressurizing the inside of the cartridge by the
pressure generation means, at least one of a point of time when the
pressure in the container is maximized and the pressure change
velocity in the container becomes zero or less and a point of time
when the pressure in the container is maximized and the pressure
change acceleration in the container is minimized, is determined as
the timing of completion of discharge of the liquid from the
container.
4. The method according to any one of the items 1 to 3, wherein,
when the pressure of the inside of the container does not reach a
set value and is not maximized in the middle of pressurizing the
inside of the cartridge by the pressure generation means, a point
of time when the pressure change velocity in the container becomes
lower than a threshold value, is determined as the timing of
completion of discharge of the liquid from the container.
5. The method according to any one of the items 1 to 4, which
comprises:
(1) injecting and pressurizing a nucleic acid-containing specimen
liquid into the container through one of the at least two openings,
and discharging the nucleic acid-containing specimen liquid from
the other opening to thereby adsorb a nucleic acid contained in the
nucleic acid-containing specimen liquid to the nucleic
acid-adsorbent solid phase;
(2) dispensing and pressurizing a washing liquid into the container
through one of the at least two openings, and discharging the
washing liquid from the container through the other opening to
remove impurities; and
(3) dispensing and pressurizing an elution liquid into the
container through one of the at least two openings to isolate and
discharge the nucleic acid adsorbed to the nucleic acid-adsorbent
solid phase from the container through the other opening together
with the elution liquid to thereby collect nucleic acid.
6. The method according to the item 5, wherein in response of the
determined timing of completion of discharge of the liquid, the
subsequent step is performed.
7. An automatic nucleic acid isolation and purification apparatus
for isolating and purifying nucleic acid from a nucleic
acid-containing specimen, the apparatus comprising:
a cartridge for isolation and purification of a nucleic acid, in
which the cartridge includes a container having at least two
openings and containing a nucleic acid-adsorbent solid phase;
a pressure generation means for generating a pressure difference
between the inside and outside of the container in the cartridge,
in which a liquid injected into the container through one of the at
least two openings is discharged from the other opening to the
outside of the container through the nucleic acid-adsorbent solid
phase by a pressure difference generated by the pressure generation
means; and
a control portion for discharging the liquid from the container on
the basis of the method according to any one of the items 1 to
6.
8. The apparatus according to the item 7, which further comprises a
means for measuring a pressure of the inside of the container.
9. The apparatus according to the item 8, wherein the control
portion comprises: an arithmetic means for calculating at least one
of a pressure change velocity and pressure change acceleration on
the basis of the value of the measured pressure; and a
determination means for determining the timing of the completion of
discharge of the liquid on the basis of the information from the
arithmetic means, in which, in response of the determined timing of
completion of discharge of the liquid, the subsequent step is
performed.
10. The apparatus according to any one of the items 7 to 9, wherein
the nucleic acid-adsorbent solid phase is a porous material
including an organic macromolecule capable of absorbing a nucleic
acid by a weak interaction not concerned with an ionic bond.
11. The apparatus according to the item 10, wherein the porous
material includes an organic macromolecule having a hydroxyl
group.
12. The apparatus according to any one of the items 7 to 11,
wherein the nucleic acid-adsorbent solid phase includes an organic
material prepared by saponifying a mixture of acetyl celluloses
different in an acetyl value.
13. The apparatus according to the item 12, wherein the
saponification rate of said mixture is not lower than 5%.
14. The apparatus according to the item 12 or 13, wherein the
organic material is a saponified mixture of triacetyl cellulose and
diacetyl cellulose.
15. The apparatus according to any one of the items 7 to 14,
wherein the nucleic acid-adsorbent solid phase is a porous film, of
which the front and back sides are asymmetrical with each
other.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic block diagram showing the configuration of
important part for automatically determining completion of a liquid
discharge step in a nucleic acid isolation and purification process
according to the invention.
FIGS. 2A to 2C show a profile in a first pattern in the case where
pressure reaches a set value within a set time, FIG. 2A being a
graph showing temporal change of pressure, FIG. 2B being a graph
showing temporal change of pressure change velocity, FIG. 2C being
a graph showing temporal change of pressure change
acceleration.
FIG. 3 is a flow chart showing-a procedure of determining
completion of the step in the case where the first pattern is
exhibited.
FIGS. 4A to 4C show a profile in a second pattern in the case where
pressure P does not reach a set value P.sub.1 within a set time
t.sub.1 but maximized within the set time t.sub.1, FIG. 4A being a
graph showing temporal change of pressure, FIG. 4B being a graph
showing temporal change of pressure change velocity, FIG. 4C being
a graph showing temporal change of pressure change
acceleration.
FIG. 5 is a flow chart showing a procedure of determining
completion of the step in the case where the second pattern is
exhibited.
FIGS. 6A to 6C show a profile in a third pattern in the case where
pressure P does not reach a set value P.sub.1 within a set time
t.sub.1 and is not maximized within the set time t.sub.1, FIG. 6A
being a graph showing temporal change of pressure, FIG. 6B being a
graph showing temporal change of pressure change velocity, FIG. 6C
being a graph showing temporal change of pressure change
acceleration.
FIG. 7 is a flow chart showing a procedure of determining
completion of the step in the case where the third pattern is
exhibited.
FIG. 8 is a flow chart showing combination of determination
algorithms shown in FIGS. 3, 5 and 7.
FIG. 9 is a perspective view showing an embodiment of a nucleic
acid extraction apparatus in a state where a cover is removed.
FIG. 10 is a schematic block diagram of the configuration of the
nucleic acid extraction apparatus.
FIG. 11 is a perspective view of a rack in a mount mechanism.
FIG. 12 is a perspective view showing a state of use of the
rack.
FIGS. 13(a) to 13 (g) are a process view showing an extracting
operation.
FIG. 14 is a perspective view of cartridge for isolation and
purification of a nucleic acid.
DESCRIPTION OF THE REFERENCE NUMERALS AND SYMBOLS
1 nucleic acid extraction apparatus (nucleic acid isolation and
purification apparatus) 2 apparatus body 3 mount mechanism 4
pressurized air supply mechanism 5 dispensing mechanism 6 rack 11
cartridge for isolation and purification of nucleic acid 11b
nucleic acid-adsorbent porous film 11c discharge portion (other
opening) 11e upper opening (one opening) 12 waste container 13
collection container 16 liquid 14 suction flow passage 18 air 40
pressurizing head 41 air nozzle 43 air pump (pressure generation
device) 45 on-off valve 46 pressure sensor 51w, 51r dispensing
nozzle 52w, 52r supply pump 56w, 56r bottle 70 control portion S
specimen liquid W washing liquid R elution liquid
BEST MODE FOR CARRYING OUT THE INVENTION
A preferred embodiment of the invention as to an method of
automatically isolating and purifying nucleic acid and the
apparatus will be described below in detail with reference to the
drawings.
FIG. 1 is a schematic block diagram showing the configuration of
important part for automatically determining the termination of a
liquid discharge step in a nucleic acid isolation and purification
process according to the invention.
This apparatus includes cartridge for isolation and purification of
a nucleic acid (hereinafter simply referred to as "cartridge") 11
containing a porous filter 11b as a nucleic acid-adsorbent solid
phase in its inside, and a pressurizing head 40 detachably attached
to the cartridge 11 so that the pressurizing head 40 can be moved
up and down by control according to detection by a position sensor
not shown. The pressurizing head 40 is connected to a pump 43
through an electromagnetic valve 45 which serves as an on-off
valve. A pressure sensor 46 is interposed in the middle of a
connected piping 74 so that pressure in the piping 74 is measured.
Measurement results of the position sensor and the pressure sensor
46 are input to a control portion 70. The pressure, pressure change
velocity and pressure change acceleration as well as positional
information of a pressurizing nozzle are calculated by the control
portion 70, so that the operations of the pump 43, the
electromagnetic valve 45 and the pressurizing head 40 are
controlled by predetermined programmed algorithm for automatically
determining the termination of a discharge step. That is, the
control portion 70 serves as arithmetic means for calculating the
pressure change velocity and pressure change acceleration and also
as determination means for determining the timing of the
termination of liquid discharge on the basis of the
information.
A pressure signal given to the control portion 7 is sampled at
intervals of a predetermined time and usually calculated as an
average value per second in order to avoid misdetermination of the
algorithm because of noise. It is preferable that the predetermined
time interval is not larger than 0.5 sec. The pressure change
velocity dP/dt obtained by differentiating pressure with respect to
time and the pressure change acceleration d.sup.2P/dt.sup.2
obtained by differentiating the pressure change velocity with
respect to time are usually calculated as average values per second
and used for the determination of the algorithm.
Next, the algorithm for automatically determining the termination
of the liquid discharge step will be described on the basis of
temporal change in pressure, pressure change velocity and pressure
change acceleration in the cartridge 11 at the time of discharge of
a liquid 16 injected into the cartridge 11 containing the nucleic
acid-adsorbent solid phase.
The temporal change in pressure, pressure change velocity and
pressure change acceleration varies widely according to the kind of
the nucleic acid-adsorbent solid phase contained in the cartridge
11 and the kind of a specimen. Accordingly, the temporal change is
classified into three typical patterns.
(First Pattern)
FIGS. 2A to 2C are graphs showing temporal change in pressure,
pressure change velocity and pressure change acceleration
respectively in a first pattern. FIGS. 2A to 2C show a profile in
the case where the pressure reaches a set value within a set time.
FIG. 3 is a flow chart showing a procedure of process termination
determination in the case where temporal change exhibits the first
pattern.
First, after a specimen containing nucleic acid is injected into
the cartridge 11, the pressurizing head 40 is attached to the
cartridge 11. Then, pressurizing the inside of the cartridge 11 by
the pump 43 starts (step 11, hereinafter abbreviated to S11).
Incidentally, the pressure is set by adjustment of the
electromagnetic valve 45 and measured by the pressure sensor 46.
After pressurizing starts, the pressure of the inside of the
cartridge 11 increases. When the pressure reaches a predetermined
set value P.sub.1 within a predetermined set time t.sub.1 (S12),
pressurizing by means of the pump 43 is stopped (S13). As a result,
the specimen in the cartridge 11 passes through the filter 11b as a
nucleic acid-adsorbent solid phase on the basis of the pressure, so
that the specimen is discharged from the cartridge 11. As the
amount of the specimen in the cartridge 11 decreases, the pressure
P of the inside of the cartridge 11 decreases from P.sub.a to
P.sub.b.
When pressurized air is extruded out of the cartridge 11 after all
the specimen is discharged from the cartridge 11, the pressure P of
the inside of the cartridge 11 decreases rapidly. On this occasion,
pressure acceleration d.sup.2P/d.sup.2 reaches a minimum a.sub.1.
For this reason, when the point of time that pressure acceleration
d.sup.2P/dt.sup.2 reaches the minimum is detected (S14) after the
pump is stopped, a decision is made that discharge is terminated,
that is, the process is terminated (S15). When pressure
acceleration d.sup.2P/dt.sup.2 does not reach the minimum within a
set time t.sub.2, a decision is made that choking error occurs
(S16). In this case, controlling is performed to operate the
electromagnetic valve 45 to open the pressure of the inside of the
cartridge 11 to the atmospheric air. The set times t.sub.1 and
t.sub.2 and the set pressure P.sub.1 can be decided at option.
Although the pressure of the inside of the cartridge 11 increases
after pressurizing starts, another determination step may be
carried out (S17) when the pressure does not reach the set value
P.sub.1 within the predetermined set time t.sub.1
(Second Pattern)
FIGS. 4A to 4C are graphs showing temporal change in pressure,
pressure change velocity and pressure change acceleration
respectively in a second pattern. FIGS. 4A to 4C show a profile in
the case where the pressure P does not reach the set value P.sub.1
within the set time t.sub.1 but reaches a maximum within the set
time t.sub.1. FIG. 5 is a flow chart showing a procedure of process
termination determination in the case where temporal change
exhibits the second pattern.
As described above in the first pattern, first, after a specimen
containing nucleic acid is injected into the cartridge, the
pressurizing head 40 is attached to the cartridge 11. When
pressurizing by means of the pump 43 then starts (S21), the
specimen in the cartridge 11 passes through the filter 11b as a
nucleic acid-adsorbent solid phase and is discharged out of the
cartridge 11. As a result, the pressure of the inside of the
cartridge 11 increases. With the passage of time, the pressure
change velocity decreases and the pressure P reaches a maximum
P.sub.C. On this occasion, the pressure velocity becomes zero and
the pressure acceleration reaches a minimum a.sub.2. For this
reason, the condition that the pressure P reaches a maximum within
the set time t.sub.1 (S22) while the pressure velocity is not
higher than zero or while the pressure acceleration reaches a
minimum is determined (S23). At the point of time that this
condition is satisfied, a decision is made that discharge is
terminated, that is, the process is terminated (S24). Therefore,
the pump 43 is stopped (S25). Incidentally, the timing of stopping
the pump 43 can be decided at option if the timing is after the
point of time that a decision is made that discharge is
terminated.
Although the pressure of the inside of the cartridge 11 increases
after pressurizing starts, another determination step may be
carried out (S26) when the pressure P does not reach the maximum
within the set time t.sub.1.
(Third Pattern)
FIGS. 6A to 6C are graphs showing temporal change in pressure,
pressure change velocity and pressure change acceleration
respectively in a third pattern. FIGS. 6A to 6C show a profile in
the case where the pressure P neither reaches the set value P.sub.1
within the set time t.sub.1 nor reaches a maximum within the set
time t.sub.1. FIG. 7 is a flowchart showing a procedure of process
termination determination in the case where temporal change
exhibits the third pattern.
As described above in the first and second patterns, first, after a
specimen containing nucleic acid is injected into the cartridge,
the pressurizing head 40 is attached to the cartridge 11. When
pressurizing by means of the pump 43 then starts (S31), the
specimen in the cartridge passes through the nucleic acid-adsorbent
solid phase and is discharged out of the cartridge 11. As a result,
the pressure of the inside of the cartridge 11 increases. In this
case, however, the pressure change velocity decreases slowly with
the passage of time. In this manner, when the pressure does not
have any maximum within the set time t.sub.1 (S32), a decision is
made that discharge is terminated, that is, the process is
terminated (S34) at the time that the pressure change velocity
reaches a set threshold P'.sub.1 or lower (S33). As a result, the
pump 43 is stopped (S35). Incidentally, the timing of stopping the
pump can be decided at option if the timing is after the point of
time that a decision is made that discharge is terminated. The
predetermined threshold P'.sub.1 of the pressure change velocity
can be set at any value.
As described above, in order to automatically determine the
termination of the process in which a liquid is injected into the
cartridge containing the filter 11b as a nucleic acid-adsorbent
solid phase and pressurized so as to be discharged, it is
preferable that a program is provided so that an optimum
determination algorithm is selected from three determination
algorithms while a determination is made as to which of the three
patterns is valid. An example of the automatic determination will
be described below.
(Automatic Determination of all Patterns)
FIG. 8 is a flow chart combined with the determination algorithms
shown in FIGS. 3, 5 and 7.
First, a determination is made as to whether the pressure P reaches
a set value P.sub.1 or higher within a set time t.sub.1 after start
of pressurizing. When the pressure P is not lower than the set
value P.sub.1, the termination of the process is determined in
accordance with the above description with reference to FIGS. 2A to
2C and FIG. 3. When the pressure P is lower than the set value
P.sub.1, a determination is made as to whether the pressure P
reaches a maximum within the set time t.sub.1. When the pressure P
reaches the maximum, the termination of the process is determined
in accordance with the above description with reference to FIGS. 4A
to 4C and FIG. 5. When the pressure P does not reach any maximum
within the set time t.sub.1, the termination of the process is
determined in accordance with the above description with reference
to FIGS. 6A to 6C and FIG. 7.
According to this configuration, the termination of the process can
be determined appropriately for any one of the first, second and
third patterns, so that the termination of the process can be
determined accurately by use of an optimum algorithm in accordance
with the fit pattern. As a result, discharge of the liquid can be
completed in the required minimum time without continuing wasteful
pressurizing in the process, so that reduction in tact time can be
attained.
As described above, the first, second and third typical patterns
have been illustrated and the determination algorithms for
determining these patterns have been described. Even if any other
pattern than these patterns is generated, the determination
algorithms can be combined suitably to determine the termination of
the process accurately and rapidly in the same manner as described
above.
Next, an example in which the aforementioned determination of the
termination of the liquid discharge step is applied to a nucleic
acid isolation and purification method will be described.
First, the nucleic acid isolation and purification method at least
includes the steps of: (1) passing a nucleic acid-containing
specimen liquid through a nucleic acid-adsorbent porous material
(nucleic acid-adsorbent porous film) to adsorb nucleic acid into
the porous film; (2) washing the nucleic acid-adsorbent porous film
in a state in which the nucleic acid is adsorbed onto the nucleic
acid-adsorbent porous film; and (3) passing an elution liquid
through the nucleic acid-adsorbent porous film to desorb the
nucleic acid from the inside of the porous film.
Preferably, in each of the steps (1), (2) and (3), the nucleic
acid-containing specimen liquid, a washing liquid, a DNase solution
or the collection solution is made to pass through the nucleic
acid-adsorbent porous film while pressurized. More preferably, in
each of the steps (1), (2) and (3), the nucleic acid-containing
specimen liquid, the washing liquid or the elution liquid is
injected into one opening of the cartridge 11 including the nucleic
acid-adsorbent porous film stored in a container having at least
two openings, and the injected solution or liquid is made to pass
through the porous film so as to be discharged from the other
opening while the inside of the cartridge 11 is pressurized by a
pressure difference generator connected to one opening of the
cartridge 11. When the nucleic acid-containing specimen liquid, the
washing liquid or the elution liquid is made to pass through the
porous film while pressurized, the apparatus can be preferably
automated compactly. Pressurizing is made preferably in a range of
from 10 to 200 kPa, more preferably in a range of from 40 to 100
kPa.
In the nucleic acid isolation and purification process, all the
procedure of from the first step of injecting the nucleic
acid-containing specimen liquid to the step of obtaining nucleic
acid in the outside of the cartridge can be completed within 10
minutes, especially within 2 minutes in a preferred condition. The
nucleic acid isolation and purification process can yield 50 mass %
or more of nucleic acid, especially 90 mass % or more of nucleic
acid in a preferred condition, with respect to the total amount of
nucleic acid contained in the specimen.
Examples of the pressure difference generator used in the
aforementioned process include: an inflatable pump such as an
injector, a pipetter or a peristaltic pump; and a deflatable
material such as an evaporator. Among these, the injector is
suitable for manual operation and the pump is suitable for
automatic operation. The pipetter has an advantage that it can be
operated easily by a single hand. Preferably, the pressure
difference generator is detachably attached to one opening of the
cartridge.
The specimen allowed to be used in this invention is not limited
but, for example, humors such as whole blood, blood plasma, blood
serum, urina, feces, semen or saliva taken as a specimen or
solutions prepared from biological materials such as a plant (or
part thereof), an animal (or part thereof), bacteria, virus or a
dissolution or homogenate thereof are used in the diagnostic
field.
First, these specimens are treated with an aqueous solution
containing a reagent (nucleic acid solubilizing reagent) for
dissolving cell membranes and nuclear membranes to thereby
solubilize nucleic acid. As a result, cell membranes and nuclear
membranes are dissolved and the nucleic acid is dispersed into the
aqueous solution, so that a nucleic acid-containing specimen liquid
is prepared.
A process of dissolving cell membranes to solubilize nucleic acid
to prepare a nucleic acid-containing specimen liquid from a
specimen will be described below. In the invention, a nucleic acid
solubilizing reagent is used for dissolving cell membranes to
solubilize nucleic acid. An example of the nucleic acid
solubilizing reagent is a solution containing chaotropic salt, and
a surfactant.
An example of the method for dissolving cell membranes to
solubilize nucleic acid to prepare a nucleic acid-containing
specimen liquid from a specimen can be a method including the steps
of: (I) injecting a cell-containing or virus-containing specimen
into a container; (II) adding a nucleic acid solubilizing reagent
solution containing chaotropic salt and a surfactant into the
container to mix the nucleic acid solubilizing reagent solution
with the specimen; and (III) adding a water-soluble organic solvent
into the mixture solution obtained by the aforementioned step.
In the process for dissolving cell membranes to solubilize nucleic
acid to prepare a nucleic acid-containing specimen liquid from a
specimen, optimization of automation is improved when the specimen
is homogenized. Homogenization can be made by a treatment such as
an ultrasonic treatment, a treatment using a high-speed agitating
treatment using a sharp-edged protrusion from fine voids or a
treatment using glass beads.
The method for mixing the nucleic acid solubilizing reagent
solution containing chaotropic salt and a surfactant with the
specimen is not particularly limited.
It is preferable that the two are mixed with each other for a time
of from 1 second to 3 minutes by an agitator rotating at a speed of
from 30 to 3000 rpm. In this manner, the yield of nucleic acid
isolated and purified can be increased. Or it is preferable that
the two are mixed with each other by repeating reversing mixture by
5 times to 30 times. Or the two may be mixed with each other by
repeating pipetting by 10 tines to 50 times. In this case, the
yield of nucleic acid isolated and purified can be increased by a
simple operation.
The nucleic acid-adsorbent porous film used in the invention and
the adsorbing process will be described below. The nucleic
acid-adsorbent porous film used in the invention can transmit the
solution through its inside. The phrase "can transmit the solution
through its inside" means that the solution can be transmitted from
a high-pressure space side to a low-pressure space side through the
inside of the film when a pressure difference is generated between
a space adjacent to one surface of the film and a space adjacent to
the other surface of the film. Or the phrase means that the
solution can be transmitted in a centrifugal direction through the
inside of the film when centrifugal force is applied to the
film.
The nucleic acid-adsorbent porous film used in the invention is
characterized to be a porous film onto which nucleic acid is
adsorbed by an interaction substantially irrespective of any ionic
bond. What is meant by this is that the porous film is not ionized
as the porous film side condition in use. It is supposed that the
nucleic acid and the porous film will attract each other when
environmental polarity is changed. As a result, nucleic acid can be
isolated and purified while both isolating performance and washing
efficiency can be kept excellent. Preferably, the nucleic
acid-adsorbent porous film is a porous film having a hydrophilic
group. It is supposed that hydrophilic groups of the nucleic acid
and the porous film will attract each other when environmental
polarity is changed. The term "porous film having a hydrophilic
group" means a porous film made of a material having a hydrophilic
group or a porous film made of a material into which a hydrophilic
group is introduced by treatment or coating. The material forming
the porous film may be an organic material or an inorganic
material. Examples of the porous film include: a porous film made
of an organic material having a hydrophilic group; a porous film
made of an organic material having no hydrophilic group but treated
so that a hydrophilic group is introduced into the porous film; a
porous film made of an organic material having no hydrophilic group
out coated with a material having a hydrophilic group so that the
hydrophilic group is introduced into the porous film; a porous film
made of an inorganic material having a hydrophilic group; a porous
film made of an inorganic material having no hydrophilic group but
treated so that a hydrophilic group is introduced into the porous
film; and a porous film made of an inorganic material having no
hydrophilic group but coated with a material having a hydrophilic
group so that the hydrophilic group is introduced into the porous
film. From the point of view of processability, an organic material
such as an organic macromolecule is preferably used as the material
for forming the porous film.
The term "hydrophilic group" means a polar group (atomic group)
which can interact with water. All groups (atomic groups)
concerning adsorption of nucleic acid are equivalent to the
hydrophilic groups. A group which can interact with water at a
medium strength (see "medium hydrophilic group" written in the item
"hydrophilic group" in Chemical Dictionary published by Kyoritsu
Shuppan Co., Ltd.) is preferred as the hydrophilic group. Examples
of the hydrophilic group include hydroxyl group, carboxyl group,
cyano group, oxyethylene group, etc. Especially, hydroxyl group is
preferred.
An example of the porous film having a hydrophilic group is a
porous film made of an organic material having a hydroxyl group. An
example of the organic material having a hydroxyl group is a
surface saponified of acetyl cellulose described in Japanese Patent
Laid-Open No. 2003-128691. Acetylcellulose can be selected from
monoacetyl cellulose, diacetyl cellulose and triacetyl cellulose.
Especially, triacetyl cellulose is preferred. In this case, the
amount (density) of the hydroxyl group in a solid phase surface can
be controlled by the degree of saponification (saponification
rate). To improve the nucleic acid isolating efficiency, it is
preferable that the amount (density) of the hydroxyl group is high.
For example, in the case of acetyl cellulose such as triacetyl
cellulose, the saponification rate (surface saponification rate) is
selected to be preferably in a range of from about 5 to 100%, more
preferably in a range of from 10 to 100%. To increase the surface
area of the organic macromolecule having a hydroxyl group, it is
preferable that the porous film of acetyl cellulose is saponified.
In this case, a porous film of which the front and back sides are
symmetrical with each other may be used as the porous film or a
porous film of which the front and back sides are asymmetrical with
each other may be preferably used as the porous film.
The term "saponification" means a treatment for bringing acetyl
cellulose into contact with a saponification solution (such as an
aqueous solution of sodium hydroxide). By this treatment, part of
the acetyl cellulose in contact with the saponification solution is
changed to regenerated cellulose so that the hydroxyl group can be
introduced into the porous film.
To change the saponification rate, the sodium hydroxide
concentration used in the saponification may be changed. The
saponification rate can be measured easily by an NMR, an IR or an
XPS. (For example, the saponification rate can be decided on the
basis of the degree of reduction in peaks of carbonyl groups).
Examples of the porous film made of an organic material having a
hydroxyl group include porous films made of polyhydroxyethyl
acrylate, polyhydroxyethyl methacrylate, polyvinyl alcohol,
polyvinyl pyrrolidone, polyacrylate, polymethacrylate,
polyoxyethylene, acetyl cellulose, mixture of acetyl celluloses
different in acetyl number, etc. Especially, a porous film made of
an organic material having a polysaccharide structure can be
preferably used.
Particularly a porous film of an organic macromolecule including a
mixture of acetyl celluloses different in acetyl value can be
preferably used as the porous film of an organic material having a
hydroxyl group. Preferable examples of the mixture of acetyl
celluloses different in acetyl number include: a mixture of
triacetyl cellulose and diacetyl cellulose; a mixture of triacetyl
cellulose and monoacetyl cellulose; a mixture of triacetyl
cellulose, diacetyl cellulose and monoacetyl cellulose; and a
mixture of diacetyl cellulose and monoacetyl cellulose.
Especially, a mixture of triacetyl cellulose and diacetyl cellulose
can be preferably used. The mixture rate (mass rate) of triaceyl
cellulose to diacetyl cellulose is preferably in a range of from
99:1 to 50:50.
A porous film made of an organic material prepared by saponifying a
mixture of acetyl celluloses different in acetyl number can be
preferably used as the porous film of an organic material having a
hydroxyl group. Preferable examples of the organic material
prepared by saponifying a mixture of acetyl celluloses different in
acetyl number include: a saponified mixture of triacetyl cellulose
and diacetyl cellulose; a saponified mixture of triacetyl cellulose
and monoacetyl cellulose; a saponified mixture of triacetyl
cellulose, diacetyl cellulose and monoacetyl cellulose; and a
saponified mixture of diacetyl cellulose and monoacetyl cellulose.
Especially, a saponified mixture of triacetyl cellulose and
diacetyl cellulose can be preferably used.
The mixture ratio (mass ratio) of triacetyl cellulose to diacetyl
cellulose is preferably selected to be in a range of from 99:1 to
50:50.
A porous film of cellulose can be preferably used as the porous
film of an organic material having a hydroxyl group. A porous film
of regenerated cellulose can be preferably used as the porous film
of cellulose. Regenerated cellulose is prepared by saponifying a
solid surface or whole of acetyl cellulose. Regenerated cellulose
is different from original cellulose in crystalline state or the
like.
As a method for introducing a hydrophilic group into the porous
film of an organic material having no hydrophilic group, a graft
polymer chain having a hydrophilic group in a polymer chain or in a
side chain can be bonded to the porous film.
Two methods may be used as the method for bonding the graft polymer
chain to the porous film of an organic material. That is, there are
a method for chemically bonding the porous film and the graft
polymer chain to each other and a method for polymerizing a
compound having polymerizable double bonds with the porous film as
a start point to thereby form a graft polymer chain.
First, in the method for attaching the porous film and the graft
polymer chain to each other by chemical bonding, a polymer having a
functional group capable of reacting with the porous film in a
terminal or side chain of the polymer is used so that this
functional group chemically reacts with a functional group of the
porous film so as to be grafted to the functional group of the
porous film. The functional group capable of reacting with the
porous film is not particularly limited if it can react with the
functional group of the porous film. Examples of the functional
group include: a silane coupling group such as alkoxysilane; an
isocyanate group; an amino group; a hydroxyl group; a carboxyl
group; a sulfonic group; a phosphoric group; an epoxy group; an
allyl group; a methacryloyl group; and an acryloyl group.
Examples of a compound especially useful as the polymer having a
reactive functional group on a terminal or side chain of the
polymer include: a polymer having a trialkoxysilyl group in a
terminal of the polymer; a polymer having an amino group in a
terminal of the polymer; a polymer having a carboxyl group in a
terminal of the polymer; a polymer having an epoxy group in a
terminal of the polymer; and a polymer having an isocyanate group
in a terminal of the polymer. The polymer used in this case is not
particularly limited if it has a hydrophilic group concerning
adsorption of nucleic acid. Specific examples of the polymer
include: polyhydroxyethyl acrylate, polyhydroxyethyl methacrylate
and salt thereof; polyvinyl alcohol, polyvinyl pyrrolidone,
polyacrylate, polymethacrylate and salt thereof; and
polyoxyethylene.
The method for polymerizing a compound having polymerizable double
bonds with the porous film as a start point to thereby form a graft
polymer chain is generally called "surface graft polymerization".
The surface graft polymerization method is a method in which an
active seed is given onto a substrate surface by means of plasma
irradiation, light irradiation, heating or the like so that a
compound having polymerizable double bonds and disposed so as to be
adjacent to the porous film is bonded to the porous film by
polymerization. The compound useful for forming the graft polymer
chain bonded to the substrate needs to have two characteristics,
that: is, the compound needs to have polymerizable double bonds and
also has a hydrophilic group concerning adsorption of nucleic acid.
Any compound such as a polymer, an oligomer or a monomer having a
hydrophilic group can be used as the compound if the compound has a
double bond in a molecule. A monomer having a hydrophilic group is
an especially useful compound.
Specific examples of the especially useful monomer having a
hydrophilic group are as follows. For example, hydroxyl
group-containing monomers such as 2-hydroxyethyl acrylate,
2-hydroxyethyl methacrylate, glycerol monomethacrylate, etc. can be
especially preferably used. Carboxyl group-containing monomers such
as acrylic acid, methacrylic acid, etc. or alkali-metal salt and
amine salt thereof can be preferably used.
As another method for introducing a hydrophilic group into the
porous film of an organic material having no hydrophilic group, the
porous film may be coated with a material having a hydrophilic
group. The material used for coating is not particularly limited if
it has a hydrophilic group concerning adsorption of nucleic acid.
From the point of view of easiness of operation, a polymer of an
organic material is preferred. Examples of the polymer may include:
polyhydroxyethyl acrylate, polyhydroxyethyl methacrylate, and salt
thereof; polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylate,
polymethacrylate, and salt thereof; and polyoxyethylene, acetyl
cellulose, and a mixture of acetyl celluloses different in acetyl
number. Especially, a polymer having a polysaccharide structure is
preferred.
After the porous film of an organic material having no hydrophilic
group is coated with acetyl cellulose or a mixture of acetyl
celluloses different in acetyl value, the acetyl cellulose or the
mixture of acetyl celluloses different in acetyl value for use in
coating may be saponified. In this case, the saponification rate is
preferably selected to be not lower than about 5%. Especially, the
saponification rate is preferably selected to be not lower than
about 10%.
An example of the porous film of an inorganic material having a
hydrophilic group can be a porous film containing a silica
compound. A glass filter can be an example of the porous film
having a silica compound. A porous silica thin film as described in
Japanese Patent No. 3,058,342 can be an example of the glass
filter. The porous silica thin film can be produced as follows.
That is, a developing solution of a cationic amphipatic substance
having the ability of forming a bimolecular film is developed on a
board. Then, a solvent is removed from a liquid film on the board
to thereby prepare a multilayer bimolecular thin film of the
amphipatic substance. The multilayer bimolecular thin film is
brought into contact with a solution containing a silica compound.
Then, the multilayer bimolecular thin film is extracted and
removed.
Examples of a method of introducing a hydrophilic group into a
porous film of an inorganic material having no hydrophilic group
include: a method of chemically bonding a porous film and a graft
polymer chain to each other; and a method of polymerizing a graft
polymer chain with a porous film as a stare point by using a
monomer having a hydrophilic group having a double bond in a
molecule.
To attach the porous film and the graft polymer chain attached to
each other by chemical bonding, a functional group which can react
with a functional group at a terminal of the graft polymer chain is
introduced into an inorganic material and the graft polymer is
chemically bonded thereto. To polymerize the graft polymer chain
with the porous film as a start point by using a monomer having a
hydrophilic group having a double bond in a molecule, a functional
group which will serves as a start point for polymerizing the
compound having a double bond is introduced into an inorganic
material. A graft polymer having a hydrophilic group and a monomer
having a hydrophilic group having a double bond in a molecule as
described in the method of chemically bonding the graft polymer
chain to the porous film of an organic material having no
hydrophilic group can be preferably used as the graft polymer
having a hydrophilic group and the monomer having a hydrophilic
group having a double bond in a molecule.
As another method for introducing a hydrophilic group into the
porous film of an inorganic material having no hydrophilic group,
the porous film can be coated with a material having a hydrophilic
group. The material used in coating is not particularly limited as
long as it has a hydrophilic group concerned with adsorption of
nucleic acid. A polymer of an organic material is preferred from
the point of view of facilitation of operation. Examples of the
polymer may include: polyhydroxyethyl acrylate, polyhydroxyethyl
methacrylate, and salt thereof; polyvinyl alcohol, polyvinyl
pyrrolidone, polyacrylic acid, polymethacrylic acid, and salt
thereof; and polyoxyethylene, actyl cellulose, a mixture of acetyl
celluloses different in acetyl value, and so on.
After the porous film of an inorganic material having no
hydrophilic group is coated with acetyl cellulose or a mixture of
acetyl celluloses different in acetyl value, the acetyl cellulose
or the mixture of acetyl cellulose different in acetyl value for
use in coating may be saponified. In this case, the saponification
rate is preferably selected to be not lower than about 5%.
Especially, the saponification rate is preferably selected to be
not lower than about 10%.
An example of the porous film of an inorganic material having no
hydrophilic group can be a porous film produced by processing a
metal such as aluminum, ceramics such as glass, cement or pottery,
or new ceramics, silicon, activated carbon, etc.
The cartridge for isolation and purification of nucleic acid is
preferably formed so that other members than the nucleic
acid-adsorbent porous film permeable to the aforementioned solution
are stored in the container having at least two openings. Plastics
such as polypropylene, polystyrene, polycarbonate, polyvinyl
chloride, etc. may be used as the material of the container. A
biodegradable material may be also preferably used. The container
may be transparent or colored.
Cartridge for isolation and purification of a nucleic acid having
means for identifying the cartridge individually can be used as the
cartridge for isolation and purification of nucleic acid. A bar
code, a magnetic tape, etc. may be used as the means for
identifying the cartridge individually.
A cartridge having a structure in which the nucleic acid-adsorbent
porous film can be easily taken out from the container having at
least two openings may be also used.
The cartridge for isolation and purification of nucleic acid in
which the nucleic acid-adsorbent porous film permeable to each
solution can be used for isolating and purifying nucleic acid by
the steps of:
(a) injecting a nucleic acid-containing specimen liquid into a
container having at least two openings, through one opening of
cartridge for isolation and purification of a nucleic acid
containing a nucleic acid-adsorbent porous film permeable to a
solution;
(b) pressurizing the inside of the cartridge for isolation and
purification of nucleic acid by using a pressure difference
generation device connected to one opening of the cartridge for
isolation and purification of nucleic acid, and passing the
injected nucleic acid-containing specimen liquid through the
nucleic acid-adsorbent porous film, and discharging the nucleic
acid-containing specimen liquid from the container through the
other opening of the cartridge for isolation and purification of
nucleic acid to thereby adsorb nucleic acid into the nucleic
acid-adsorbent porous film;
(c) injecting a washing liquid into the container through one
opening of the cartridge for isolation and purification of nucleic
acid;
(d) pressurizing the inside of the cartridge for isolation and
purification of nucleic acid by using the pressure difference
generation device connected to one opening of the cartridge for
isolation and purification of nucleic acid, and passing the
injected washing liquid through the nucleic acid-adsorbent porous
film, and discharging the washing liquid from the container through
the other opening to thereby wash the nucleic acid-adsorbent porous
film in a state in which nucleic acid is adsorbed on the nucleic
acid-adsorbent porous film;
(e) injecting an elution liquid into the container through one
opening of the cartridge for isolation and purification of nucleic
acid; and
(f) pressurizing the inside of the cartridge for isolation and
purification of nucleic acid by using the pressure difference
generation device connected to one opening of the cartridge for
isolation and purification of nucleic acid, and passing the
injected elution liquid through the nucleic acid-adsorbent porous
film, and discharging the elution liquid from the container through
the other opening to thereby desorb nucleic acid from the nucleic
acid-adsorbent porous film and discharge the nucleic acid from the
container of the cartridge for isolation and purification of
nucleic acid.
In another embodiment, nucleic acid may be isolated and purified by
the steps of:
(a) injecting a nucleic acid-containing specimen liquid into a
container having at least two openings, through one opening of
cartridge for isolation and purification of a nucleic acid
containing a nucleic acid-adsorbent porous film permeable to a
solution;
(b) reducing the pressure of the inside of the cartridge for
isolation and purification of nucleic acid by using a pressure
difference generation device connected to the other opening of the
cartridge for isolation and purification of nucleic acid, and
passing the injected nucleic acid-containing specimen liquid
through the nucleic acid-adsorbent porous film, and discharging the
nucleic acid-containing specimen liquid from the container through
the other opening of the cartridge for isolation and purification
of nucleic acid to thereby adsorb nucleic acid into the nucleic
acid-adsorbent porous film;
(c) injecting a washing liquid into the container through one
opening of the cartridge for isolation and purification of nucleic
acid;
(d) reducing the pressure of the inside of the cartridge for
isolation and purification of nucleic acid by using the pressure
difference generation device connected to the other opening of the
cartridge for isolation and purification of nucleic acid, and
passing the injected washing liquid through the nucleic
acid-adsorbent porous film, and discharging the washing liquid from
the container through the other opening to thereby wash the nucleic
acid-adsorbent porous film in a state in which nucleic acid is
adsorbed on the nucleic acid-adsorbent porous film;
(e) injecting an elution liquid into the container through one
opening of the cartridge for isolation and purification of nucleic
acid; and
(f) reducing the pressure of the inside of the cartridge for
isolation and purification of nucleic acid by using the pressure
difference generation device connected to the other opening of the
cartridge for isolation and purification of nucleic acid, or
applying centrifugal force on the cartridge for isolation and
purification of nucleic acid, and passing the injected elution
liquid through the nucleic acid-adsorbent porous film, and
discharging the elution liquid from the container through the other
opening to thereby desorb nucleic acid from the nucleic
acid-adsorbent porous film and discharge the nucleic acid from the
container of the cartridge for isolation and purification of
nucleic acid.
In a further embodiment, nucleic acid may be isolated and purified
by the steps of:
(a) injecting a nucleic acid-containing specimen liquid into a
container having at least two openings, through one opening of
cartridge for isolation and purification of a nucleic acid
containing a nucleic acid-adsorbent porous film permeable to a
solution;
(b) applying centrifugal force on the cartridge for isolation and
purification of nucleic acid, and passing the injected nucleic
acid-containing specimen liquid through the nucleic acid-adsorbent
porous film, and discharging the nucleic acid-containing specimen
liquid from the container through the other opening of the
cartridge for isolation and purification of nucleic acid to thereby
adsorb nucleic acid into the nucleic acid-adsorbent porous
film;
(c) injecting a washing liquid into the container through one
opening of the cartridge for isolation and purification of nucleic
acid;
(d) applying centrifugal force on the cartridge for isolation and
purification of nucleic acid, and passing the injected washing
liquid through the nucleic acid-adsorbent porous film, and
discharging the washing liquid from the container through the other
opening to thereby wash the nucleic acid-adsorbent porous film in a
state in which nucleic acid is adsorbed on the nucleic
acid-adsorbent porous film;
(e) injecting an elution liquid into the container through one
opening of the cartridge for isolation and purification of nucleic
acid; and
(f) applying centrifugal force on the cartridge for isolation and
purification of nucleic acid, and passing the injected elution
liquid through the nucleic acid-adsorbent porous film, and
discharging the elution liquid from the container through the other
opening to thereby desorb nucleic acid from the nucleic
acid-adsorbent porous film and discharge the nucleic acid from the
container of the cartridge for isolation and purification of
nucleic acid.
The steps for isolating and purifying nucleic acid from a nucleic
acid-containing specimen by using the cartridge for isolation and
purification of nucleic acid including a container having at least
two openings and a nucleic acid-adsorbent porous film stored in the
container, and the pressure generation means may be preferably
performed by an automatic apparatus for performing the steps
automatically. In this manner, nucleic acid of a predetermined
level can be obtained regardless of the operator's skill as well as
the operation can be simplified and quickened.
The steps for isolating and purifying nucleic acid from a nucleic
acid-containing specimen by using the cartridge for isolation and
purification of nucleic acid and the pressure generation means will
be described below with reference to an automatic nucleic acid
extraction apparatus.
As shown in FIGS. 9 and 10, the automatic nucleic acid extraction
apparatus 1 includes an apparatus body 2, a mount mechanism 3, a
pressurized air supply mechanism 4, and a dispensing mechanism 5.
The mount mechanism 3, the pressurized air supply mechanism 4 and
the dispensing mechanism 5 are disposed in the apparatus body 2.
The mount mechanism 3 holds a plurality of cartridges 11, a waste
container 12 and a collection container 13. The pressurized air
supply mechanism 4 introduces pressurized air into the cartridge
for isolation and purification of nucleic acids 11. The dispensing
mechanism 5 dispenses a washing liquid W and an elution liquid R
into the cartridges 11. The mechanisms 3 to 5 will be described
below specifically.
<Mount Mechanism>
The mount mechanism 3 has a mount table 21 in a front lower portion
of the apparatus body 2. A rack 6 in which the plurality of
cartridges 11, the waste container 12 and the collection container
13 are held is placed on the mount table 21. As also shown in FIG.
4, the rack 6 has a stand 61, a cartridge holder 62, and a
container holder 63.
The stand 61 holds the cartridge holder 62 in pillar portions 61a
on opposite sides so that the cartridge holder 62 can move up and
down. The stand 61 holds the container holder 63 on a bottom plate
61b in a lower portion between the pillar portions 61a so that the
container holder 63 can move back and forth.
The cartridge holder 62 is formed as a split-half structure in
which front and rear plates are joined to each other. The cartridge
holder 62 has a holding portion 62a extending horizontally, and
support legs 62b extending vertically and disposed at opposite ends
of the holding portion 62a. The support legs 62b are inserted into
vertically extending slide grooves 61c of the pillar portions 61a
of the stand 61 so that the support legs 62b can move up and down.
The support legs 62b are urged upward by urging members (not shown)
included in the stand 61. In the holding portion 62a, a plurality
of holding holds. 62c are provided side by side. The cartridges 11
are inserted into the holding holes 62c from above. Lower ends of
protrusions 11d (see FIG. 14) formed on opposite sides of
cylindrical bodies 11a of the cartridges 11 are fitted and held
into fitting members (not shown) in the cartridge holder 62. The
fitting members are movable. To move the fitting members, fitting
to the protrusions 11d is released so that all the cartridges 11
can be dropped down and discarded simultaneously.
The cartridge holder 62 has pin holes 62d on opposite sides of its
upper surface. In a condition of use, the cartridge holder 62 is
pushed down while front ends 49a of pressure pins 49 (see FIG. 9)
as alignment means which will be described later are fitted into
the pin holes 62d respectively. In the position where the cartridge
holder 62 is moved up as shown in FIG. 11, lower ends of discharge
portions 11c of the cartridges 11 held in the cartridge holder 62
are located above the waste container 12 and the collection
container 13 set in the container holder 63. When the cartridge
holder 62 is moved down as shown in FIG. 12, setting is made so
that the discharge portions 11c of the cartridges 11 are inserted
by a predetermined value into the waste container 12 or the
collection container 13.
The container holder 63 has waste container holding holes 63a and
collection container holding holes 63b extending horizontally and
arranged in two parallel rows. A plurality of waste containers 12
are held in the waste container holding holes 63a provided on the
rear side so that the plurality of waste containers 12 can be
arranged in a row. A plurality of collection containers 13 are held
in the collection container holding holes 63b provided on the front
side so that the plurality of collection containers 13 can be
arranged in a row. The waste container holding holes 63a and the
collection container holding holes 63b are arranged in positions
equal to those of the holding holes 62c of the cartridge holder 62
and at equal intervals of a pitch equal to that of the holding
holes 62c. The waste container holding holes 63a and the collection
container holding holes 63b are set so that the waste containers 12
and the collection containers 13 are located below the held
cartridges 11 respectively. To prevent confusion, it is preferable
that the waste containers 12 are different in size, shape, etc.
from the collection containers 13.
The container holder 63 is urged forward by an urging member not
shown but included in the stand 61. The movement (back and forth
movement) of the container holder 63 for exchanging the containers
is performed in the condition that an operation member 31 (see FIG.
10) set on the mount table 21 is fitted into a fitting hole (not
shown) of a bottom portion of the container holder 63 through an
opening formed in the bottom plate 61b of the stand 61. The
container holder 63 is moved back in accordance with the moving
operation of the operation member 31 in accordance with the drive
of a container exchange motor 32 (DC motor) so that the collection
containers 13 are located below the cartridge holder 62. At the
time of non-operation, the container holder 63 is urged by the
urging member not shown so that the waste containers 12 are located
below the cartridge holder 62. The operation of the container
exchange motor 32 is controlled in accordance with detection by
position sensors 33a and 33b.
<Pressurized Air Supply Mechanism>
As shown in FIGS. 9 and 10, the pressurized air supply mechanism 4
has a pressurizing head 40, a plurality of air nozzles 41, an air
pump 43, a relief valve 44, on-off valves 45, and pressure sensors
46. The pressurizing head 40 can move up and down relative to the
rack 6 of the mount mechanism 3. The plurality of air nozzles 41
(eight air nozzles in FIG. 9) are arranged in a row in the
pressurizing head 40. The air pump 43 generates pressurized air.
The on-off valves 45 are disposed in the air nozzles 41
respectively so that the air nozzles 41 can be opened and closed
individually. The pressure sensors 46 are set in the air nozzles 41
respectively. Pressurized air is supplied to the cartridges 11
successively. The air pump 43, the relief valve 44 and the air
nozzles 41 operate on the basis of a control instruction given from
a control portion 70.
The pressurizing head 40 is held by guide rods 24 disposed
vertically between an intermediate frame 22 and an upper frame 23
of the apparatus body 2 so that the pressurizing head 40 can move
up and down. A ball nut 40a disposed in the pressurizing head 40 is
thread-engaged with a ball screw 25 set vertically in the same
manner as the guide rods 24. The pressurizing head 40 is controlled
in accordance with detection by photo sensors 48a to 48c so that
the pressurizing head 40 are moved up and down in accordance with
the rotation of the ball screw 25 in accordance with the drive of
an elevation motor 47 (pulse motor) through a timing belt and a
pulley. Pressure pins 49 are provided on opposite sides of the
pressurizing head 40. The pressure pins 49 are urged downward by
springs 49b so that the pressure pins 49 can move up and down.
Front ends 49a of the pressure pins 49 are fitted into the pin
holes 62d in the upper surface of the cartridge holder 62 so that
the pressure pins 49 presses the cartridge holder 62 while the
positions of the front ends 49a are limited.
The pressure pins 49 are disposed to press the front position of
the cartridge holder 62 so that the movement of the pressure pins
49 does not interfere with the horizontal movement of a washing
liquid dispensing nozzle 51w and an elution liquid dispensing
nozzle 51r (which will be described later) in the condition that
the pressure pins 49 operate to press the cartridge holder 62.
The air nozzles 41 are disposed in the pressurizing head 40 so that
the air nozzles 41 can move up and down individually while urged
downward. A sheet-like sealing material 42 in which connection
holes 42a (see FIG. 10) corresponding to the air nozzles 41 are
opened is disposed below the air nozzles 41. When the pressurizing
head 40 moves down, upper end openings of the cartridges 11 set in
the cartridge holder 62 are pressed and closed by the front ends of
the air nozzles 41 through the sealing material 42 so that
pressurized air can be supplied into the cartridges 11 through the
connection holes 42a.
The relief valve 44 is operated to open atmospheric air when air is
discharged from passages between the air pump 43 and the on-off
valves 45. An air circuit is formed so that the on-off valves 45
are selectively operated to be opened so that pressurized air can
be introduced from the air pump 43 into the cartridges 11 through
corresponding air nozzles 41. That is, suction flow passages 14 are
formed between the air pump 43 and the cartridges 11. Pressurized
air is supplied on the basis of an instruction given from the
control portion 70. The pressure sensors 46 are disposed in the air
nozzles 41 respectively and detect the pressures of the inside of
the cartridges individually. The pressure sensors 46 cooperate with
the control portion 70 to perform controlling for determining
completion of discharge of liquid on the basis of the
aforementioned algorithm in accordance with a pattern of pressure
change.
<Dispensing Mechanism>
The dispensing mechanism 5 has a washing liquid dispensing nozzle
51w, an elution liquid dispensing nozzle 51r, a washing liquid
supply pump 52w, an elution liquid supply pump 52r, and a waste
bottle 57. The washing liquid dispensing nozzle 51w and the elution
liquid dispensing nozzle 51r are disposed in a nozzle movement
table 50 which can move horizontally on the rack 6. The washing
liquid supply pump 52w supplies a washing liquid W stored in a
washing liquid bottle 56w to the washing liquid dispensing nozzle
51w. The elution liquid supply pump 52r supplies an elution liquid
R collected into an elution liquid bottle 56r to the elution liquid
dispensing nozzle 51r. The waste bottle 57 is placed on the mount
table 21.
The nozzle movement table 50 is held by a guide rail 27 set
horizontally on a vertical wall 26 of the apparatus body 2 so that
the nozzle movement table 50 can move horizontally. The movement of
the nozzle movement table 50 is stopped on the cartridges 11
successively by a nozzle movement motor (pulse motor) not shown.
For restoration, the drive of the nozzle movement table 50 is
controlled so that the nozzle movement table 50 stops on the waste
bottle 57. Each of the washing liquid dispensing nozzle 51w and the
elution liquid dispensing nozzle 51r has a front end bent downward.
The washing liquid dispensing nozzle 51w is connected to the
washing liquid supply pump 52w through a change-over valve 55w. The
washing liquid supply pump 52w is connected to the washing liquid
bottle 56w through the change-over valve 55w. The elution liquid
dispensing nozzle 51r is connected to the elution liquid supply
pump 52r through a change-over valve 55r. The elution liquid supply
pump 52r is connected to the elution liquid bottle 56r through the
change-over valve 55r. The washing liquid bottle 56w and the
elution liquid bottle 56r are disposed on a side of the apparatus
body 2. The washing liquid supply pump 52w and the elution liquid
supply pump 52r are formed as syringe pumps respectively. Pump
motors 53w and 53r (pulse motors) control to drive piston members
of the syringe pumps so that a predetermined amount of washing
liquid W and a predetermined amount of elution liquid R can be
dispensed on the basis of position detection by sensors 54w and 54r
respectively. These pump motors 53w and 53r and change-over valves
55w and 55r operate on the basis of an instruction given from the
control portion 70.
To dispense the washing liquid W or the elution liquid R, the
change-over valve 55w or 55r is switched to the washing liquid
bottle 56w side or the elution liquid bottle 56r side to drive the
pump motor 53w or 53r to move back the piston member of the washing
liquid supply pump 52w or the elution liquid supply pump 52r. As a
result, the washing liquid W or the elution liquid R is sucked and
stored into the washing liquid supply pump 52w or the elution
liquid supply pump 52r. Succeedingly, the change-over valve 55w or
55r is switched to the washing liquid dispensing nozzle 51w side or
the elution liquid dispensing nozzle 51r side to drive the motor
pump 53w or 53r to push in the piston member of the washing liquid
supply pump 52w or the elution liquid supply pump 52r. After the
washing liquid or the elution liquid is discharged from the washing
liquid dispensing nozzle 51w or the elution liquid dispensing
nozzle 51r to the waste bottle 57 until air in the passage is
exhausted, the drive of the washing liquid supply pump 52w or the
elution liquid supply pump 52r is stopped. Then, the washing liquid
dispensing nozzle 51w or the elution liquid dispensing nozzle 51r
is moved on the cartridges 11. While the quantity of drive of the
washing liquid supply pump 52w or the elution liquid supply pump
52r is controlled, a predetermined amount of the washing liquid W
or the elution liquid R is dispensed into each cartridge 11.
Each of the washing liquid bottle 56w and the elution liquid bottle
56r has a container body 56wb or 56rb, and a cap 56wu or 56ru. Slim
pipe-like suction tubes 58w and 58r are disposed in the caps 56wu
and 56ru respectively. Lower ends of the suction tubes 58w and 58r
are opened near bottom portions of the container bodies 56wb and
56rb so that the washing liquid W or the elution liquid R can be
sucked up in accordance with the operation of the washing liquid
supply pumps 52w worth elution liquid supply pump 52r.
The mechanisms 3 to 5 are controlled to be driven on the basis of a
program stored in advance in a storage portion 72 connected to the
control portion 70, by the control portion 70 associated with an
input operation of an operation panel 7 disposed in the upper
portion of the apparatus body 2.
An operation of extraction by the nucleic acid extraction apparatus
1 will be described specifically. First, the cartridges 11 are set
in the cartridge holder 62 in the rack 6 of the mount mechanism 3.
The waste containers 12 and the collection containers 13 are set in
the container holder 63 in the rack 6 of the mount mechanism 3. The
rack 6 is placed on the mount table 21 of the apparatus body 2 to
perform preparation. Then, a specimen liquid S subjected to
dissolving treatment is injected into the cartridges 11
successively by a pipette or the like. Incidentally, the specimen
liquid S may be injected into the cartridges 11 after or before the
cartridges 11 are set in the rack 6 before the rack 6 is mounted in
the apparatus 1.
When the apparatus is then operated by the operation of the
operation panel 7, the pressurizing head 40 moves clown by the
drive of the elevation motor 47 of the pressurized air supply
mechanism 4 so that the front ends 49a of the pressure pins 49 are
fitted into the pin holes 62d of the cartridge holder 62 and press
and move down the cartridge holder 62 to limit the position of the
cartridge holder 62. At the same time, a lower end discharge
portion 11c of a cartridge 11 is inserted by a predetermined
quantity into a waste container 12 as shown in FIG. 12 so that
contamination with the discharged liquid leaked out by scattering
or the like can be prevented. The pressurizing head 40 further
moves down so that the lower end portion of each air nozzle 41
comes into pressure contact with the upper end opening of the
cartridge 11 through the sealing material 42 to thereby close the
upper end opening of the cartridge 11. On this occasion, because
the position of the cartridge holder 62 is limited by the pressure
pins 49, the air nozzles 41 can come into pressure contact with the
cartridges 11 respectively accurately to attain sure closure.
Then, pressurized air is supplied. The air pump 43 is driven on the
basis of an instruction given from the control portion 70 in the
condition that all the on-off valves 45 are closed. The first
on-off valve 45 is opened. A predetermined amount of pressurized
air is supplied from the air pump 43 to the first cartridge 11
through the first air nozzle 41.
Then, the first on-off valve 45 is closed and the second on-off
valve 45 is opened. Pressurized air is supplied to the second
cartridge 11 through the second air nozzle 41. This operation is
repeated successively so that pressure is applied on all the
cartridges 11. When the specimen liquid S on which pressure acts
passes through the nucleic acid-adsorbent porous film 11b, nucleic
acid is adsorbed and held on the nucleic acid-adsorbent porous film
11b. Other liquid components than nucleic acid are discharged from
the discharge portion 11c of the lower portion to the waste
container 12. When all the specimen liquid S passes through the
nucleic acid-adsorbent porous film 11b, pressure change as shown in
FIGS. 2A to 2C , 4A to 4C and 6A to 6C occurs. When the pressure
change is detected, completion of discharge of liquid is
determined. In this manner, when completion of extraction in all
the cartridges 11 is detected by the pressure sensors 46, the
pressurizing head 40 is moved up.
Then, processing is shifted to washing treatment. The up movement
of the pressurizing head 40 after supply of the pressurized air is
performed while the state shown in FIG. 12 is kept, that is, while
the state where the air nozzle 41 is departed from the cartridge 11
and stopped in the position where the air nozzle 41 is moved up to
a height allowing the nozzle movement table 50 to move, the
pressure pins 49 press the cartridge holder 62 and the lower end of
the cartridge 11 is inserted into the waste container 12. Then, the
nozzle movement table 50 is moved. The washing liquid dispensing
nozzle 51w is stopped on the first cartridge 11. A predetermined
amount of the washing liquid W is dispensed into the first
cartridge 11. The nozzle movement table 50 is moved to the next
cartridge 11 so that the washing liquid W is dispensed into the
cartridges 11 successively. When dispensing of the washing liquid W
into all the cartridges 11 is completed, the pressuring head 40
moves down. After the lower end portion of each air nozzle 41 comes
into pressure contact with the upper end opening of a corresponding
cartridge 11 through the sealing material 42 to close the upper end
opening, the on-off valves 45 are operated successively in the same
manner as described above. Thus, pressurized air is supplied into
the cartridges 11 in the same manner as described above. The
washing solution W on which pressure acts passes through the
nucleic acid-adsorbent porous film 11b to thereby wash and remove
other impurities than nucleic acid. The washing liquid W is
discharged from the discharge portion 11c of the lower portion to
the waste container 12. When the washing liquid W in all the
cartridges 11 is discharged after passage through the nucleic
acid-adsorbent porous film 11b, the pressurizing head 40 is moved
up to the initial position. When the washing treatment needs to be
performed several times, the aforementioned operation is
repeated.
Then, processing is shifted to collecting treatment. First, the
pressure pins 49 move up in accordance with the up movement of the
pressurizing head 40 after the washing treatment, so that the
cartridge holder 62 of the rack 6 also moves up. After the lower
end discharge portion 11c of the cartridge 11 moves up with respect
to the waste container 12, the operation member 31 of the mount
mechanism 3 is operated to move back the container holder 63. The
collection container 13 located below the cartridge 11 is
exchanged.
Then, the pressurizing head 40 moves down. The front ends of the
pressure pins 49 are fitted into the pin holes 62d of the cartridge
holder 62 to press the cartridge holder 62. In this manner, the
state where the lower end of the cartridge 11 is inserted into the
collection container 13 is kept. Then, the nozzle movement table 50
is moved. The elution liquid dispensing nozzle 51r is stopped on
the first cartridge 11. A predetermined amount of the elution
liquid R is dispensed into the first cartridge 11. The nozzle
movement table 50 is moved to the next cartridge 11 so that the
elution liquid R is dispensed into the cartridges 11 successively.
When dispensing of the elution liquid R into all the cartridges 11
is completed, the pressuring head 40 moves down in the same manner
as described above. After the lower end portion of each air nozzle
41 is brought into pressure contact with the upper end opening of
the cartridge 11 through the sealing material 42 to close the upper
end opening, the on-off valves 45 are opened successively. Thus,
pressurized air is supplied into the cartridges 11 successively in
the same manner as described above. The elution liquid R on which
pressure acts passes through the nucleic acid-adsorbent porous film
11b to thereby desorb nucleic acid adsorbed on the nucleic
acid-adsorbent porous film 11b. Thus, nucleic acid is discharged
together with the elution liquid R from the discharge portion 11C
of the lower portion to the collection container 13. When the
elution liquid R in all the cartridges 11 is discharged to the
collection containers 13, the pressurizing head 40 is moved up.
Thus, a series of operations is completed.
After the extracting operation is completed, the rack 6 is removed
from the mount table 21. The cartridges 11 and the waste containers
12 are taken out from the cartridge holder 62 and the container
holder 63 and discarded. On the other hand, the collection
containers 13 are taken out from the container holder 63, covered
if necessary, and subjected to the next nucleic acid analyzing
treatment.
Although this embodiment has shown the case where a plurality of
cartridge for isolation and purification of nucleic acids 11 are
mounted, the invention is not limited thereto. For example, the
invention may be applied to the case where only one cartridge for
isolation and purification of nucleic acid 11 is mounted.
Any gas may be used as air supplied from the air pump 43 to the
cartridge for isolation and purification of nucleic acid 11 as long
as the gas has no influence on the property of the liquid 16.
INDUSTRIAL APPLICABILITY
According to the invention, when a liquid is injected into a
container having a nucleic acid-adsorbent solid phase stored
therein and is discharged from the container through the nucleic
acid-adsorbent solid phase by a pressure difference generated
between the inside and outside of the container, completion of
discharge of the liquid can be automatically determined accurately
and rapidly regardless of the difference in kind of the nucleic
acid-adsorbent solid phase stored in the container, the difference
in production lot of the nucleic acid-adsorbent solid phase, the
difference in production lot of the container per se, the
difference in the container in each production lot, the kind of the
liquid to be used, and so on. Accordingly, in automation of the
nucleic acid isolation and purification steps using the nucleic
acid-adsorbent solid phase excellent in isolating performance,
efficiency can be improved so greatly that take time can be
shortened and that the throughput of isolation and purification can
be improved greatly.
* * * * *